Our goal has been to define the important molecular events that control T cell maturation within the thymus. After migrating to the thymus, hematopoietic precursor cells undergo a complex set of developmental events and emerge as mature T lymphocytes capable of specific antigen recognition. This developmental process appears to result from a series of signalling interactions between T cell precursors and a heterogeneous and as yet ill-defined set of stromal cells. We are studying three areas of thymic development at the molecular level. First, we have established a culture system in which day 14/15 triple negative fetal thymocytes can be stimulated to express early markers of thymocyte development including CD25, ICAM-1, and Ly-6A/E. We have found that IL-1 and TNF are critical mediators of progression through the first stage of thymic development leading to expression of the CD25 surface marker. Blockade of these cytokines prevents further maturation of thymocytes to the double positive stage. Our in vitro culture system has allowed us to identify a precursor cell in the thymus that gives rise to T cells, B cells, and natural killer cells. This has allowed us to determine that commitment to the T lineage occurs at the CD25+ stage. Second, we have discovered that the block to the development of double positive T lymphocytes in mice deficient for the recombinase-activating gene (RAG) can be overcome by irradiation. This appears to involve the p53 molecule. Third, we are creating genetically-engineered mice that are homozygous-deficient (knocked-out) for various genes that are highly expressed in thymocytes. We are currently focusing on three genes: i) Ly-GDI - a GDP-dissociation inhibitor protein that controls the activity of Rho, a member of the Ras family that may be involved in molecular signalling cascades; ii) Rlk - a newly described member of the btk/itk family of tyrosine kinases that have been recently implicated in inherited immunodeficiency states and are involved in signalling from surface antigen receptors; and iii) Ntk - a newly-described tyrosine kinase related to the Csk kinase that regulates various Src family kinases by phosphorylating inhibitory tyrosine residues. Homozygous deficient mice have been constructed for LyGDI and Rlk and work is underway on the Ntk knock-out. Since signalling interactions are required for proper T cell differentiation, disruption of these signalling molecules may inhibit T cell develop in ways that clarify the normal physiological roles of these molecules.